Abstract The ability to infect non-dividing cells is what separates lentiviruses, including HIV-1, from other retroviruses which require the breakdown of the nuclear envelope during cell division to access the genome of target cells. Lentiviruses, in contrast, have evolved the ability to selectively translocate their genome across the nuclear pore complex (NPC). Unfortunately, the mechanism by which HIV-1 translocates through the NPC remains one of the least well understood aspect of the viral lifecycle, in large part because there is no direct, definitive method to monitor HIV-1 nuclear import. In this application, we describe a method to monitor HIV-1 nuclear import kinetics directly through the ability to selectively and potently inhibit HIV-1 nuclear import at various times post-infection. This method prevents infection of any viruses which have not yet traversed the NPC and entered the nucleus, and thereby allows us to directly monitor the kinetics of nuclear import by measuring the fraction of the viral inoculum which has cleared the NPC at times following a synchronized infection. As such, this assay provides a unique opportunity to appreciate nuclear translocation of the viral genome in target cells, including primary cells, and to determine how specific viral determinants and cellular factors influence the nuclear translocation of the viral genome.